Abstract
Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt‐1 and Flk‐1. Yet, their relative importance for hypoxia‐induced hyperpermeability is not well understood. We used an in vitro blood‐brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt‐1 in VEGF‐induced endothelial cell (EC) barrier dysfunction. Soluble Flt‐1 abolished hypoxia/VEGF‐induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt‐1, but not to Flk‐1, inhibited hypoxia‐induced permeability changes. Consistent with these data, addition of the receptor‐specific homolog placenta‐derived growth factor, which binds Flt‐1 but not Flk‐1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF‐E, a viral VEGF molecule from the orf virus family activating Flk‐1 and neuropilin‐1, but not Flt‐1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt‐1, it was demonstrated that activation of Flt‐1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3‐kinase/Akt (PI3‐K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3‐K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF‐induced hyperpermeability can be mediated by activation of Flt‐1 independently on the presence of Flk‐1 and indicate a central role for activation of the PI3‐K/Akt pathway, followed by induction of NOS and PKG activity. J. Cell. Physiol. 212: 236–243, 2007. © 2007 Wiley‐Liss, Inc.